Method for sedimentation study

ABSTRACT

Method for the study of the sedimentation characteristics of whole blood comprising the steps of cyclically applying greater than gravity force laterally to a thin, substantially vertically oriented column of whole blood, and rotating the column about 180 degrees about its own axis between each cycle. The sample columns are placed in tubes arranged with their long axes oriented substantially parallel to the axis of rotation of a centrifuge and the tubes are rotated about their own long axes between each cycle and only when at or substantially at rest. A preferred test operation using four cycles of 45 second duration is described with the rotation of the columns being effected by reversal of the direction of rotation of the centrifuge head at the end of each cycle. Other cycle durations are described for providing results correlatable with standarized sedimentation test procedures.

United States Patent 1191 Bull 1451 July 23, 1974 [54] METHOD FORSEDIMENTATION STUDY 3,695,842 10/1972 Mintz 73/64.] X

[75] Inventor: Brian S. Bull, Loma Linda, Calif.

Primary ExaminerRichard C. Queisser [73] Ass1gnee: Coulter Electronlcs,lnc., H1aleah, Assistant Examiner joseph w Roskos Attorney, Agent, orFirmSilverman & Cass [22] Filed: Oct. 22, 1971 [21] Appl. No.: 191,886[57] ABSTRACT Related U.S. Application Data Method for the study of thesedimentation characteris- [63] Continuation-impart of s 13 1 3 tics ofwhole blood comprising the steps of cyclically 1971, abandoned. Iapplying greater than gravity force laterally to a thin, substantiallyvertically oriented column of whole [52] U.S. Cl. 73/61.4, 23/230 B,73/64.l blood, and rotating the column about 180 degrees [51] Int. Cl.G0ln 15/04, G0ln 33/16 about its ownaxis between each cycle. The sample[58] Field of Search 73/61.4, 64.1, 61 R; columns are placed in tubesarranged with their long 23-3/26j23/230 B axes oriented substantiallyparallel to the axis of rotation of ,a centrifuge and the tubes arerotated about [56] References Cited their own long axes between eachcycle and only when UNITED STATES PATENTS at or substantially at rest. Apreferred test operation using four cycles of 45 second duration isdescribed 52 :I:I'"'::::"""""""':::: with the rotation of the columnsbeing effected by re- 3:009:388 11/1961 Polanyi 73/61.4 Verse of thedirection of rotation of the Centrifuge 3,026,717 3/1962 Danielsson etal... 73/61.4 head at t end of each y other Cycle durations 3,373,6013/1968 Monn 73/61.4 are described for providing results correlatablewith 3,460,752 8/1969 Lucas 233/26 standarized sedimentation testprocedures. 3,503,709 3/l970 Yochem 73/64.l X 3,518,057 6/1970 Giordano73/64.1 X 25 Claims, 12 Drawing Figures 60 .SEC.

FIG. 2

22 INVENTORY E M BRIAN s. BULL ATT'YS 1 METHOD FOR SEDIMENTATION STUDYCROSS-REFERENCE TO RELATED APPLICATION This application is acontinuation-in-part of my copending application, Ser. No. 113,166,filed Feb. 8, 1971, entitled SEDIMENTATION RATE TEST METHOD ANDSEDIMENTATION RATE CENTRI- FUGE THEREFOR now abandoned.

FIELD OF THE INVENTION This invention relates generally to diagnosticexami-' nation of whole blood and more particularly concerns theprovision of improved and means for whole blood sedimentation study.

1 BACKGROUND OF THE INVENTION of clinical analysis. Usingthesedimentation test results,

the presence of more or less occult disease can be brought to medicalattention. Such results particularly are of importance in thedifferential diagnosis as between functional disorders having closelysimilar symptomatic manifestations, as well as in supplying a guide tothe progress of certain diseases. Accordingly, it is believed thatsubstantial benefit could be obtained in the diagnosis and treatment ofmedical disorders by the establishment of sedimentation study procedureswhich would produce comparative information quickly and economically sothat a sedimentation study could become a routine procedure in clinicalexamination. However, as practiced presently, the sedimentation test istoo time consuming, too affected by laboratory introduced artifacts andsubject to misinterpretation in anemic individuals, so that the test isnot a test offered to every patient as a routine clinical test proceduresuch as a blood count, for example. 1

Present methodology involves essentially the mixing of a whole bloodsample with a selected anticoagulant, introducing this well mixed samplein a vertically arranged glass tube and permitting the red cells of thesample to sediment under the influence of gravity. This process is slow,usually taking sixty or more minutes. The only accepted variations inthis method takes the form, singly or in combination, merely of changingthe length of the glass tubes employed, varying the bore of such tubes,careful selection of the anticoagulant employed and/or modification ofthe degree of dilution utilized. None of these variations havealleviated the principal drawback to adoption of the sedimentation testas a routine procedure, this drawback being that present sedimentationrate tests methods are too time consuming for routine employment or massstudies.

Another important deterrent to adoption of sedimentation testing as aroutine procedure has been the extreme sensitivity of this test to thearrangement of the test sample in an absolutely vertical orientation forthe duration of the test. It has been found that a sample column whichis oriented at only a three degree offset from vertical will result ininconsistent acceleration of the sedimentation rate and reduces therelative differences between the comparative normal and abnormal bloodsedimentation characteristics, thereby reducing the value of the test indiagnosis.

Accordingly, it is the principal object of the invention to provide animproved sedimentation study method for whole blood which meets therequirements for rapidity, economy, accuracy and reliability essentialfor adoption as a mass applied clinical laboratory test procedure, and,concomitant therewith, to provide a sedimentation rate centrifugeparticularly adapted for implementing and carrying out the steps of theimproved study method.

Another object of this invention is to provide an improved sedimentationstudy method which provides comparative information on the sedimentationbehavior of whole blood from normal persons and from persons sufferingfrom functional disorders, this information being provided quickly andwith reliability, the method being free from the sensitivity of priormethods to dispostion of the test samples during performance thereof;and also which results can be obtained approximately equivalent tostandard methods of sedimentation testing and also can provide asedimentation study result independent of hematocrit effect of thesample.

SUMMARY OF THE INVENTION A sedimentation study method for whole bloodcom prising the steps of applying greater than gravity force less than8.25 G laterally to a substantially vertically oriented column of wholeblood sample in a repeated series of applications and rotating thecolumn about its own vertical axis between each application of force;thereafter determining the concentration of cells in the resultingpacked portion of said sample. According to the subject method, acomparison is made between the start level and the treated level. Thecolumn may be then fully packed by centrifugation at 100 G or the likeand a comparison again made to the treated column level. A ratio then isdetermined of the two results to provide a hematocrit independent value.A centrifuge apparatus is provided for implementing the subject method,comprising a centrifuge head and a motor, the centrifuge head carryingat least a pair of sample tube holders arranged to orient the samples insubstantially vertical columnar array, drive means connected be- BRIEFDESCRIPTION OF THE DRAWINGS FIG. 1 is a diagrammatic representationillustrating an improved sedimentation rate study method according tothe invention;

FIG. 2 is a perspective view of the sedimentation rate centrifugeconstructed in accordance with the invention; v

FIG. 3 is a vertical section taken through the centrifuge of FIG. 2along the lines 3-3 and in the direction indicated;

FIG. 4 is a vertical section taken through line 44 of FIG. 2 and in thedirection indicated;

FIG. 5 is a top plan view of the centrifuge arrangement as shown in FIG.2;

FIG. 6 is a vertical section taken through a modified embodiment oftheinvention; I

FIG. 7 is a perspective view of a further modified embodiment of theinvention;

DESCRIPTION OF THE PREFERRED EMBODIMENTS The method of studying thesedimentation characteristics of whole blood in accordance with theinvention, capitalizes in part upon the fact that the blood from anormal, healthy individual has greater suspension stability than doesblood from a sick individual. Three phases are known to occur during thesedimentation of wholeblood. The first is characterized as the phase ofrouleaux formation. During this phase, the red cells of the whole bloodstack together in what is defined in the art as rouleaux. This phaseoccupies the first few minutes subsequent to filling of thesedimentation tube with sample. Next begins the phase of maximumsedimentation wherein after about three to five minutes, the red cellrouleaux reach their maximum velocity of fall. This velocity is dictatedby theaverage density of the rouleaux and the viscosity of the plasmathrough which they are falling. The last phase concerned ischaracterized as the, packing phase. As the rouleaux reach the bottom ofthe sedimentation tube, they pack and, as a consequence, the averagerate of fall decreases and eventually, when packing is complete, nofurther change, occurs.

The samples from both normal and sick individuals, given enough time,will pack to approximately the same extent; but the blood samples fromthe ill patient goes through the rouleaux formation phase and into thephase of maximum velocity of fall more rapidly than does the blood froma healthy individual. An isolated red cell is so small a particle thateven though its density is considerably greater than that of the plasma,the large relative surface area becomes the over-riding factor and anisolated red cell is plasma falls very slowly under the influence ofgravity. The rate of fall of red cells is thus governed almost entirelyby thesize of the rouleaux which they form. The'blood from a healthyindividual forms such rouleaux much more slowly than does blood from asick person. If both samples are set up simultaneously, there is aperiod of time when application of force greater than gravity to theblood will affect the blood from a healthy individual minimally and thatfrom a diseased patient maximally. The crucial or critical time isobvious when the healthy blood has just only-begun to form rouleaux andthe sick blood has formed large rouleaux which have already begun tosediment.

It had been critical to the study of sedimentation rate that theconventional test must be performed under conditions where thesamplecolumn is disposed in absolutely vertical orientation. A tilt aslittle as three degrees from vertical under gravity will accelerate thesedimentation rate considerably, and decrease the relative differencesbetween normal and abnormal blood. It is believed that this effect isdue to the fact that red cells falling through the plasma hit the wallsof the container and roll down the walls permitting the plasma freeegress from the depth of the sample. Whenever the plasma is forced totraverse the descending column of red cells, the sedimentation rate isslowed. Normal red cell rouleaux, fall much more slowly than do abnormalred cell rouleaux, probably because the forces holding them together areweaker and the upsweeping plasma either breaks them up or prevents themfrom forming large enough clumps to sediment rapidly. Accordingly, byapplying a greater than gravity force to the sedimenting blood,according to the invention, the ascending plasma is forced to traversethe descending red cells. As will be explained, a slight inclination ofthe column up to about 6 from vertical is permissible with the method ofthe invention, particularly to avoid spilling of the sample during therun.

According to the invention, greater than gravitational force is appliedlaterally to a long thin column of whole blood sample by rotationthereof in a centrifuge capable of delivering a force in the range of 2to 12 G with the test taking from one-half hour at a minimum G to aboutone minute at the high end of the aforesaid range. The higher the GForce, the shorter the elapsed time of the test. The net effect is toforce the red cells to traverse the plasma component of the blood over avery short distance since the effective cross-sectional area of the tubeis now vast relative to the wall surface area and the red cells cannotcollect in one portion of the tube against the wall so as to permit theplasma to escape freely elsewhere. Insofar as the sedimentation processis concerned, the long thin column has been transformed to a shallowwide diameter pool and since the force is directed substantiallyperpendicular to the long axis of the column, the problems of channelingheretofore experienced in sedimentation testing'are obviated. g

The laterally applied force acts to pack the red cells rapidly,permitting the plasma physically to change location therewith andapproach the final packing state over a much shorter time period, thannormally would be expected under gravitational force.

One example utilizing the method according to the invention, permits thecompletion of the packing stage under gravity subsequent to the periodicapplication of the greater than gravity force and the level of thepacked red cell rouleaux observed and compared with that of a normalblood standard sample treated under the same conditions.

Another example of the subject method involves the obtaining of apacking factor after the said periodic cycles and a second packingfactor on.maximum packing by centrifugation under more than G. The ratioof these factors is a value indicative of the presence of asymmetricprotein, a fact important to state of health evaluations and independentof hematocrit.

The selection'of the duration of the centrifugation cycles as well astheir number are dependent also upon the speed of the drive orsynchronous spinner motor utilized, the diameter of the column and ofthe sedimentation tube utilized, and the degree of cant or tiltpermitted.

Another method of practicing the invention involves a program selectedto use four cycles of applied force laterally to the column and of45second duration each. Between the first and second, the second and thirdand the third and fourth cycle of centrifugation, the columns arerotated about their own vertical axes. Care is taken to assure that theaxial rotation of the columns take place only when the force appliedlaterally to the long axes of the columns is less than one G. Thiscondition occurs when the column isat rest, substantially at rest or, toput it another way, begins its translation in its circumferentialrotation with and on the centrifuge head, that is, to the spool on whichthe columns ride,

at the time each cycle is ending so that a gradual braking is effectedso that the centrifuge head may be stopped without abrupt rotation ofthe columns. Abrupt stoppage of the columns must be avoided, andlikewise, jarring or other sharp disturbance of the columns are avoidedso that the cells which have separated from the plasma and gathered atthe inner wall of the tube containing the test sample column will not bebroken awayfrom each other or from the tube wall. The cells in thecolumnmust rotate with the tube, and the column as the same is rotatedin accord with the invention.

In accordance further with the method of the invention, between eachapplication of greater than gravity force, the test sample column isrotated about its own long axis, preferably 180.

This seems to provide beneficial results in obtaining reproduciblepacking in alternatively permitting resuspending the cells by rotatingthe tube and its column contents and again effectively forcing thosecells back through the plasma. Thus, according to the invention,cohesive forces are utilized during the centrifugation under therelatively low G force to force the cells against the wall anddispersion forces are utilized when the cells are forced back throughthe plasma by again centrifuging but after 180 rotation of the tube andcol umn of test sample.

Clearly, the rotation of the tube containing the'test sample column onits axis must be sufficiently gradual so as not to sever the adhesionbetween the column of cells and the tube wall. Sharp rotation will leavethe cells, while the wall moves. Since the purpose generally of theaforesaid column rotation is to effectively force the cells through theplasma component of the sample so as to provide a reasonablyreproducible packing factor, movement of the cells with tube wall duringsaid tube rotation on its axis is mandatory.

Now referring to the diagrammatic flow chart of FIG. 1, attention isdirected first to the sedimentation test method according to theinvention. Following this method will provide test results equivalent tothe Wintrobe standard method of sed.rate testing. The blood sample S istaken from the patient by means of a syringe 12.01" the like,transferred to avessel and mixed with any one of a plurality of selectedanticoagulants. A predetermined amount of mixed sample is placed in along, thin tube, sometimes referred to in the art as a microhematocrittube 16 and which constitutes the test sample. The tube 16 is filled toa standardized depth. The steps involved in the production of this'testsample are well known in the art and is represented by box 14 in FIG. 1.The microhematocrit tube 16 generally of 2 'mm diameter is placedsubstantially vertically in one of the tube holders 46 of the centrifuge20. The other holders are likewise employed .with other samples so thatthe centrifuge is balanced. As will be discussed later, otherconfigurations of tube holders are contemplated.

The range of speed of rotation of the centrifuge 20 preferably isselected between 200 and 1,000 revolutions per minute so that aneffective centrifugal force of approximately 7.25 is provided. The motor22 of the centrifuge 20 is energized and the centrifuge is caused tooperate, here in a clockwise direction, for a relatively short spin, sayabout 20 seconds. The centrifuge then is stopped and brought to rest.When the centrifuge is at rest or at least substantially at rest, thetube holder 18 and its tube 16 arecaused to rotate about its own longaxis and the centrifuge 20 again is caused to rotate, applying a forceof approximately 6.25 to 8 G to the vertically oriented column of testsample in tube 16. This second rotation also is for a short duration,again about 20 seconds, after which the centrifuge 20 is brought to arest condition and the tube holder 18 and the tube 16 therein again arerotated 180 about its own axis. A reversing motor is used so that thecentrifuges direction of rotation is changed with each cycle. Therotation of the test column about its own axis may be in a directionopposite to the immediately preceding direction of rotation of thecentrifuge 20 because it is easier to effect rotation of the tube andtube holder about their own long axes due to the inertia of thecentrifuge head in starting. A unidirectional centrifuge also can beused with the direction of rotation of the tubes and tube holders abouttheir own axes remaining unchanged from cycle to cycle.

The first two short spins are for the purpose of accelerating rouleauxformation by shunting the red cells back and forth through the plasma tocause them to collide without really effecting the sedimentationthereof. During these spins, the red cells are either isolated or atmost in groups of two to four cells and as a result they are not movedany appreciable distance by the approximately 6.25 to 8 G force appliedperpendicular to the long axis of the tubes during the first two shortspins. The cells are moved further than they would move under gravityinfluence during this period and collisions between individual cells forthe formation of rouleaux accelerated. After the pair of short spins,the rouleaux formation in blood from an ill person has taken place to alarger extent than it would if the blood sample originated from a normalor healthy person.

After the two short spins of about 20 seconds each and the rotation ofthe test column about its own vertical axis each thereafter, therouleaux formation is such that the approximately 6.25 to 8 G force maybe applied when the test sample is experiencing the second phase ofsedimentation, that is the maximum velocity of descent of the formedrouleaux in the test sample from the ill person, a time whereat thesedimentation rate can be most effectively accelerated and compared tothe reaction of the standard or normal test sample wherein rouleauxformation is barely initiated.

The centrifuge 20 is caused to rotate again to apply a force ofapproximately 6.25 to 8 G perpendicular to the long or vertical axis ofthe upstanding sample column, but this time, the application of said Gforce laterally to the columnis continued for a period of about 60seconds, a long enough period of time to move the red cell rouleauxphysically to one side of the tube. After this longer centrifugation,the tubes and tube holders once again are brought to rest condition, andthe said tubes and tube holders again are rotated 180 about their ownaxes. Two more 20 second spins follow with intermediate rotation of thetubes and tube holders about their own vertical axes. These final twospins are intended to aid the red cell column to re-establish itself inits vertical tube so that its level can be read. The rouleaux layer ispermitted to fall free of plasma hindrance. The plasma component ispermitted to escape from the red cell rouleaux column held against thetube wall. No hindrance to such passage can be expected since the cellcolumn is held against the wall of the tube by the approximately 6.25 to8 G force while the l G natural gravitational force applied axiallycauses them to travel to the bottom of the tube. The tubes, having beenfilled to a standard depth, the level of the packed cells is read bycomparison to a fixed scale either mounted on the centrifuge-head asshown in FIGS. 2, 6 and 8 for example, thereby being compared to thelevel of the packed cells observed in a standard or nor- TIME (Minutes)APPROXIMATE FORCE in G's l lO-l2 Preferably the range of G force appliedin the course of operating the particular embodiment is approximately6.25 to 8 G's with a generally preferred force application of 7.25 G.

One of the substantive disadvantages of conventional sedimentation testmethods is the criticability'of vertical orientation of the test column.With the method of the invention the slight inclination of the column ispermissible. Inclination of the tubes from the vertical such that thelower end of the tube is further from the axis of rotation of thecentrifuge head introduces a component of force up the tube andtherefore in opposition to the gravitational force which is tending tomove the particles down the tube. It thus slows the sedimentationprocess and increases the-amount of time required to perform the test.Provided the inclination of the tube from the vertical is not excessive,there is no effect upon the test other than the increased time requiredordinarily. Excessive can best be defined in the following manner. Ifthe rotation of the centrifuge head is subjecting the tubes to ahorizontal acceleration of 8 G, there will be a component of this forceup the tube of 8 G times tangent theta where thetais the inclinationbetween the tubes and the vertical. In this example, as the angle thetaapproaches 6, the upward component approaches 1 G. At 1 G there iseffective neutralization of the gravitational force as long as thecentrifuge is spinning and during this time period, which occupiesslightly more than half of theusual cycle, the'cells will move neitherup nor down the tube; they will of course move outwards. For remainderof the cycle the cells are under I G and will, of course, traveldownwards; the net effect is simply to prolong the time required toperform the test. The time gets shorter and shorter as the tubes arereturned more and more to the vertical position and indeed continues toshorten if the tubes are inclined with their top ends inwards. But withfurther inclination beyond about 6 instability and irreproducibilitybecome a problem. The effective limits therefore are from vertical withan inward or outward cant of approximately 6 if the force applied is inthe range of 8 G's. Thus one can state that the greaterthan gravityforce (relative centrifugal force) is applied to a column inclined atanangle theta from the vertical such that the relative centrifugal forceapplied times the tangent of the angle theta is a value equal to or lessthan I.

The cycles set forth as an example in FIG. 1 give results in terms of asedimentation rate correlative with and equivalent to the value obtainedby following the well known Wintrobe method of sedimentation ratedetermination. A result approximately equivalent to other standardmethod values can be obtained by variation of the number and duration ofthe cycles.

One problem which is encountered in interpreting sedimentation rateinforamtion as applied to medical diagnosis is the effect that'thehematocrit has upon the sedimentation rate and the difficulty inascribing the effect of an anaemic condition upon the observed value. Itis extremely difficult to apply corrections to observed sedimentationrates to correct for the effect of the hematocrit thereupon. An anaemicblood sample may be observed to have a certain sedimentation rate whichotherwise may indicate an abnormal functional condition or which may bedue to the anaemia condition. Correction for the effect of hematocritupon the sedimentation rate observed was not possible routinely usingconventional sedimentation rate testing methods. However, it has beenobserved that the effect of hematocrit in the method according to theinvention is a linear one and a hematocrit correction chart can beconstructed. A simple mathematical correction to all observedsedimentation rates observed following the method of the invention.whereby all sedimentation rates can be reported at a standard or normalhematocrit, say for example, 45 percent. Thus heretofore experiencedmisinterpretation of the results in anaemic individuals may beeliminatedQ In lieu of the standardized sedimentation rate valuesobtained, say pursuant to the method outlined in FIG. 1, one followinganother example of such method may obtain information independent of thehematocrit factor. Here, the cycles are of substantially of equalduration; in one example, 45 seconds under the 6.25 8 G applied in fourcycles with rotation of the tubes and columns about their own verticalaxes between each cycle. A ratio of initial to packing level is taken.The tubes may then be packed to a maximum extent by application of Gforce of at least G and a ratio of the first and maximum packing levelstaken. The ratio of the resultant maximum packing factor and the firstpacking factor is taken with the resultant value, here termed a ZSR, avalue independent of hematocrit fluctuations. i

In summary, in following the method of the invention as described above,the red cell sedimentation has been accelerated by increasing thegreater than gravity force applied at the time when abnormal blood wouldbe most sensitive to the multi G effect than blood from a normal healthyperson, this time being during the maximum velocity phase ofsedimentation reached prior to the time it would be reached if thesample were from a normal healthy person. Further, the multi G force isapplied laterally to a thin vertically oriented column of blood sampleto obviate the problems of channelizing occurring where sedimentationdoes not act absolutely parallel with the walls of the vessel containingthe sample column; the container effectively being transformed from thelong thin vertically arranged tube to one which has a wide diameter. Theintermittent rotation of the column about its own vertical axis betweenthe centrifugal spins is intended to hold the red cells on one side ofthe column while permitting the plasma to move on the other side sothat, in effect, the red cells are repeatedly forced to move through theplasma with a reproduced packing factor being the ultimate result.

The performance of the method above described required a centrifugecapable of applying the preferably G force in the range of 6.25 to 8 Gin separate cycles of predetermined duration. The centrifuge is requiredto rotate the tube containing the sample in a circumferential path aboutthe axis of rotation with the tube being in substantially verticallyoriented disposition parallel to the axis of rotation of the centrifugehead although a cant from vertical of up to 6 is permitted, contrary tostandard sedimentation rate methods. Additionally, the centrifuge isrequired not only to permit rotation of the tube along saidcircumferential path for a predetermined length of time and then thetube periodically must be brought at least to a momentary substantiallyrest or stationary condition, then rotated page, the contents of thesample tube having a tendency to remain stationary while the tube andtube holder rotate. This must be avoided. According to the method of theinvention, the column contents of the sample tube must rotate axiallywith the tube wall and care must be taken to assure only such rotation.

The centrifuge constructed in accordance with the invention andillustrated in FIGS. 1 to 5 comprises a motor 22, generally one which isreversible, for rotating a head 24 in the range of 200 to 1,000 R.P.M.Generally, the speed of rotation can be varied easily by selection ofheads of different diameter. The effective force output preferred is inthe range of 6.25 to 8 'G. The shaft of the motor 22 is coupled to thehead 24 by fastening means 28.

The head 24 comprises a spool mounted for rotation on a shaft32; Theshaft 32 has an enlarged end 34 having a passage'36 to receive the shaft26 of the motor 22. A spur wheel 38 having circumferential teeth 40 ismounted at the opposite end of shaft 32 coaxial with the spool 30. Alocking ring 41 is fastened to the shaft about its own axis apredetermined number of degrees,

and the rotation of the tube along said circumferential path resumed forthe next cycle of greater than G force application. At least, therotation of the tube about its axis must not occur during theapplication of greater than G force.

The centrifuge also should have timing means T for selectivelycontrolling and/or varying the duration of the cycles. According to themethod of FIG. 1, the duration of the successive cycles are not equalbut follow a definite program. Another example of the subject method hassuccessive equal duration cycles. The greater the G force, the shorterthe cycles and total elapsed time. In addition to the means required torotate the individual test sample holders between cycles when thecentrifuge is brought to a substantially rest condition, the centrifugemust be provided with means whereby the tube holder is brought to restor at least substantially to rest in its travel along itscircumferential path before the rotation of the tube holder about itsvertical axis can take place. This feature is required so that thecontents of the sample tube carried by the tube holder, that is, thecolumn itself, will, rotate with the rotation of tube and tube holder.Too rapid angular acceleration or rotation centrifugally tends to resultin slip- 32 by fastening means, such as screw 42. The spur wheel 38rotates with the shaft 32. The spool 30 carries a plurality of openings44 circumferentially disposed to receive tube holders 46. The tubeholders 46 are arranged in diametrically opposed pairs for balance, onlyone pair being shown in the FIGS. l-6 for convenience. Each of the tubeholders 46 is provided with a pinion wheel 48 either securedfrictionally or otherwise thereto or integral therewith. The body ofholder 46 may be transparent so that the tube 16 may be viewed and agraduated scale 52 mounted on the spool 30 adjacent the tube holder forreference in reading. The openings 44 are so arranged that the pinionwheels 48 mesh with the circumferential teeth 40 of the spur wheel 38.Each tube holder 46 is constructed with a top opening bore 50 capable ofreceiving in vertical orientation, the microhematocrit tube 16 whichcontains the sample of blood to be tested. Each tube holder 46 may havea leaf spring 51 within the bore as an aid in maintaining the properdisposition of the tube. An upstanding pin 54 is secured to the upperdisc 56 of the spool and a slot 58 is provided in the spur wheel 38.Slot 58 is configured in the form of a segment of an are, as shown inFIG. 5. The length of the slot is selected to assure that the rotationof the pinion wheel 48 is taken through exactly The spool 30 is mountedto the shaft 32 so that it is freely rotatable, the spur wheel 38 beingsecured so that it rotates with shaft 32. After the pin 54 reaches theend of slot 58, no further rotation of the pinion wheel 48 can takeplace. The motor 22 is brought to rest and then started in the reversedirection. At this time, the pinion wheel 48 must first rotate, owing tothe inertia of the spool 30, until the pin 54 reaches the other end ofthe slot 58. Thus, the carrier tube and hence the microhematocrit tube16 is caused to rotate exactly 180 with each reversal of the drivingmotor 22.

Deceleration of the motor 22 before it stops, has the same effect on thespool 30 as the reversal of the motor, so that the 180 rotation of thetube 16 takes place before the tube 16 comes to rest. The tubes must beat rest before rotation about their own axes to avoid rotation only ofthe tubes rather than the column. This problem can be overcome byintroducing a constant drag on the spool 30 such as, for example, amagnetic induction brake or a friction pad (not shown).

A preferred method of alleviating the said deceleration effect is theprovision of an interlocking device designated generally by referencecharacter 60 (FIG. 4). The interlocking device 60 comprises a metalstrip 61 pivotally secured to a block 62 which, in turn, is attached tothe inside wall of disc 56 of the spool30. The strip 61 is pivoted as at64 to move in radial slot 66. A weight or mass 68 is provided at thelower end of strip 61 and secured thereto. When the spool 30 isrotating, the mass 68 is held radially outward of the spool 30 so thatthe strip 61 engages the spur wheel 38. This condition remains until themotor speed falls to zero. At this time, the mass 68 falls to its restposition and the strip 61 disengages from the wheel 38. It should benoted that the teeth of the spur wheel between the correct engagementpositions are omitted as indicated at 70 in FIG. 5. In lieu of the pinand slot arrangement specifically illustrated in FIGS. 2, 4 and 5, onecan utilize a spoked wheel gear instead of wheel 38, with a pair of pinsdisposed between the spokes so as to limit the rotation of the piniongear 48 and with it, the sample tube and test column therewithin.

In FIG. 6, there is illustrated a sedimentation rate centrifuge whichhas been modified so as to obviate the need for a reversing motor,utilizing instead a magnetic solenoid electrically interlocked so thatthe 180 rotation of the tubes can be brought about entirely by the same.The centrifuge 20 incorporates a motor 22 and a head 24'. The motor 22'drives the shaft 32 by means of reduction gears 72 and 74. The shaft 32carries a pair of spaced discs 76 and 76 which are secured thereto forrotation therewith. A helical splined portion 78 is likewise fixed tothe shaft 17 to rotate therewith. A spur wheel 80 engages with thesplined portion 78 of the shaft 17 so that longitudinal movement of thespur wheel 80 with respect to the splined portion 78, causes the spurwheel 80 to rotate with respect to the shaft 17 by an amount sufficientto rotate pinion wheels 48 through 180. The pinion wheels 48 eachcarries a chuck or holder 46 into which the lower end of amicrohematocrit tube 16 can be inserted. Aligned openings82 are providedin disc 76 so as to support the microhematocrit tube 16 in verticalorientation, parallel to the rotational axis'of shaft 17.

The lower face of spur wheel 80 carries a thrust race 84.which can beurged upwards by means of the lever 86 mounted for vertical pivotablemovement about shaft 88. The thrust race 84 comprises a receptor ring 90secured to the lower face of spur wheel 80 and a lower ring 92 havingprotrusions 94 arranged for engagement with shallow recesses 96 formedin ring 90. The end 98 of lever 86, remote from the race 84, is operatedby means of solenoid 101. When the solenoid 101 is not energized, thespur wheel 80 is urged downward by light, annular spring 102, arrangeddisposed between disc 76 and the spur wheel 80.

The assembly consisting of the shaft 32' with its splined portion 78,the discs 76 and 76 and the pinions 48 rotate together in suitablebearings (not shown) while the motor 22, the solenoid 101 and the lever86 remain stationary.

Energization of the solenoid will rotate the tubes 52 through 180whether the motor 22' is running or not tion of the centrifuge 20'.

A further modified embodiment of the centrifuge apparatus according tothe invention is illustrated in FIG. 7 and designated by referencecharacter 100. Apparatus as described herein particularly is adapted topractice the method of the invention where the cycle utilized comprisesfour cycles of 45 second duration applications of greater thangravityforce laterally to substantially vertically arranged columns of wholeblood with means provided to effect limited rotation of each columnabout its own axis between each force application by reversal of thedirection of rotation of the centrifuge head after each cycle.

The centrifuge apparatus 100 includes a-housing 102, including atroughlike portion 104 and a cover 106. Wall 108 of housing 102 carriesexterior accessible switch levers 110 and 112 for activating the powerand buzzer means respectively which will be described hereinafter.Indicator lights 114 and 116 likewise are provided. Start switch 118 forinitiating each test operation is provided.

The electrical control components of apparatus 100 are mounted withinthe troughlike portion while the head, centrifuge 120, drive means 122,and the timing means 124 are mounted on the cover portion, thecentrifuge head being removably mounted to the protruding portion of themotor drive shaft 154 of means 122. The drive means -122 and timer means124 are mounted to be enclosed within the housing 102 when the cover 106is engaged'onto the portion 104.

The centrifuge head comprises a spool formed by mounting a spinner plate126 fixedly secured to the shaft 128 for rotation therewith, andmounting a disc 130 to the upper end of said shaft 128 with the disc 130arranged coaxial with said spinner plate 126 and. being rotatabletherewith. The shaft 128 is secured to the motor drive shaft 154 as bysuitable means such as set screw 127.

Gear support means 132 is arranged secured to the cover 106 and includesa collar portion 192 having a flat upper surface 192, and acircumferential flange portion which is fastened to the cover 106. Thespur gear 134 has a circumferential ring portion 134 carryingcircumferential teeth 134" and a central disc portion 135 to which it isfixedly mounted and by which the spur gear is mounted for independentrotation about the shaft 128, that is, independent of rotation of thespinner plate 126. A coating or film of thin machine oil or vacuum pumpoil is applied to the surface 192 of collar portion 192 to provide afriction drag upon the disc portion 135 which rests thereupon, andwhich, of course is transferred to the ring gear 134 and thereby isapplied directly to the spinner plate 126.

Tube holders 136, similar to holders 46, are mounted on spinner plate126 for movement therewith about the axis of shaft 128. The holders arespaced circumferentially substantially equidistant one relative to theother closely adjacent the peripheral edge of the spinner plate 126.Each tube holder 136 has a top opening cavity 137 defined therein toreceive the lower end of sample 'tube and has resilient means forgripping said tube seated therein, such as O-ring 139. Each holder ismounted on the upper end of a shaft 138 which extends through suitableopenings formed in said plate 126. Pinion gears 140 fixedly are securedto the opposite ends of each shaft 138 thereby mounting the holders 136on plate 126. The holders 136 are rotatable with rotation of gears 140.

A spur gear in the form of ring gear 134 is arranged so that its teeth134" are meshed with plural pinion gears 140. Thus, rotation of theplate 126 will effect rotation of gears 140 while the ring gear 134remains stationary, rotating holders 136 about their own vertical axesindependent of the rotation of the shaft 128. Limit means in the form ofthe upstanding pin 142 secured to the support means 132 and movablewithin the limit slot 144 formed in the spinner plate 126 is-provided tolimit the independence of movement of the spinner plate 126 and gear134, thereby limiting the degree of rotation of the gears 140 abouttheir own axes. The limit means described may be said to comprise a lostmotion coupling between the plate 126 and gear 134.

The disc 130 has a plurality of bottom opening recesses 146 formedequispaced about the peripheral edge thereof and arranged in alignmentwith the axes of the holders 136 but slightly offset inwardly therefromso that one end of the sample tube 150 can be seated within cavity 137of holder 136 and the upper end retained within the respectivelymatching recess 146 to position the tube substantially verticallyarranged but canted inwardly at its upper end toward the shaft 128.Thus, when properly seated, tubes 150 are disposed,

, canted inwardly from true vertical between 3 and 6,

preferably by 3 and generally not more than about 6. In thisdisposition, the tendency for the contents of the tube to be flungoutward during the application of higher than gravity force on rotationor spinning of the centrifuge head 120 materially is reduced.

Motor mount 152 is secured to the undersurface of cover 106 with thedrive shaft 154 thereof protruding through a suitable opening formed insaid cover 106. The drive means 122 for the apparatus 100 is supplied bya 400 RPM, 60 HZ, 115 volt AC reversible direction motor 156. Here,motor 156 causes centrifugal force between 66 and 8 G to be appliedlaterally to the tubes 150 during the spin of header 120. The particularsize and RPM drive motor selected determines the centrifugal forceexerted on the tubes 150, and thereby is an important factor inselection of the duration of greater than gravity application cycle andprogram.

The method of the invention requires application of the greater thangravity force laterally and periodically to the sample in the tube i.e.,the sample tube 150 and the column of blood therein. The duration ofeach cycle generally can be selected to provide results correlative withspecifically known blood sedimentation methods.

With apparatus 100, timer means 124 is provided to effect rotation ofthe spinner plate automatically through a sequence of four cycles of 45seconds duration with the reversal of direction and rotation of thecolumns 180 about their own axes between each application of centrifugalforce.

In the apparatus 100 illustrated, the timer motor 156 is an AC 60 cycle,1 volt motor delivering RPH.

The timing means 124 operates switch means, 184,186 which operatesrelays 184" and 186" automatically taking the sample columns through theselected test program.

A timing means 124 comprises a timer motor 158 mounted on platform 159,which in turn is secured suspended below the cover 106 by means of bolts160 and spacers 162. The resultant drive shaft 164 of motor 158 ispassed through a suitable opening in the platform 159 and wheel gear 166is mounted at the free end thereof for rotation therewith. A secondwheel gear 168 is coupled to gear 166 and is driven thereby. Gear 168 isfixedly secured to shaft 170 for rotation therewith. One end of shaft170 is seated in journal 172 and the other end carries timer disc 176.Timer disc 176 is secured to shaft 170 and continuously rotatestherewith so long as timer motor 156 operates through the complete testprogram. The timer disc 176 has three paddle assemblies 178, 178' and178" mounted thereto about the periphery thereof with the paddles 180,180' and 180" extending outwardly from the circumferential edge thereofin vertical planes normal to the axis of shaft 170. As illustrated, disc176 is rotatable in the direction of arrow 177 with the paddleassemblies 178, 178' and 178" fixed in an equispaced series along saidpath. An upstanding pin 182 is secured normal to the disc 176 androtates therewith. The paddle assemblies 178, 178 and 178", whenconsidering the direction of rotation of the disc, can be said to besubstantially equispaced one relative to the others with paddle assem'blies 180 and 180" being disposed 180 apart. A pair of push-buttonactivated switches 184 and 186 are arranged with their actuators 184'and 186' mounted to suitable bracket means (not shown) secured totheplatform 159 so that the actuator 184' of switch 184 is arranged inthe path of travel of the paddles 180, 180' and 180" of paddleassemblies 178, 178' and 178" whereby each respective paddle can engageand depress said actuator 184' by engaging same during passage therepastduring rotation of the disc 176.

The actuator 186' of switch 186 is positioned to intercept the pin 182whereby the continuing rotation of disc 176 causes pin 182 first to bearagainst actuator 186' to depress same. On passing of said pin 182 pastactuator 186', said actuator returns to its normal condition. The switch184 is connected to relay assembly 184" which is electrically coupled tothe reversible synchronous drive motor 156 to cause reversing of thedirection of said motor each time the actuator 184' is depressed. Theswitch 186 is connected to relay assembly 186" operatively coupledelectrically to both the drive motor 156 and to the buzzer means 190.Depression of the actuator 186 energizes the buzzer 190 and release ofthe actuator 186' from engagement with the pin 182, causesde-energization of the drive or spinner motor 156.

A friction or other drag is applied to the centrifuge head so thatapplication of braking force to'the motor 156 on de-energization of thesame, causes a braking force to be applied directly to the head.Accordingly, the tubes and the columns of test samples therein will beprevented from being rotated about their own vertical axes at leastuntil the centrifuge head 120 starts up after coming to a substantiallyfull stop, however momentary.

The friction drag described may be applied by means of the engagement ofthe collar portion 192 of gear support means 132 with the facing surfaceof gear 134 and the provision of a coating or film of light machine vhead 120 is spun.

oil sandwiched therebetween. Instead of the collar portion 192 being anupstanding ring integral with the support means 132, it may take theform of a foam collar (not shown) secured thereto or even arrangedcoaxial about the shaft 128. This oil interface friction dragarrangement is illustrated in detail in FIG. 12.

An example in testing operation utilizing apparatus 100 now will bedescribed. Samples of whole blood are taken and placed respectively inclosed end, elongate tubes known as sedimentation tubes. The tubes arefilled with sample to a predetermined level mark. The tubes containingthe test samples are placed between the disc 130 and the spinner plate126, the lower ends of the tubes seated within the tube holders 136while the upper ends are seated in the recesses 146 and held firmly bythe resilient means 139. The switch levers 110 and 112 are actuatedrespectively activating the apparatus 100. The start toggle switch 118is actuated initiating thetest procedure and causing the spinner motor156 to operate in one direction, say clockwise. Greater than gravityforce in the range of 6.25 to 8 G is applied to the column of sample ineach tube as the centrifuge When motor 156 is energized to spin head120, motor 158 is energized simultaneously to rotate disc 176. Timerdisc 176 rotates to bring paddle 180 in contact with the actuator 184.Disc 176 continues to rotate so as to carry paddle 180 past saidactuator 184. In passing, the paddle 180 depresses the actuator 184,causing the spinner motor 156 to reverse direction. This occurs 45seconds after initiation of the spinner operation.

In reversing direction, the centrifuge head 120 comes to a momentaryhalt with the pin 142 at one end of the opening or slot 144. Thecentrifuge head 120 then begins to rotate in the clockwise direction.The gear 134,

being mounted for free rotation about the shaft 128, will remainstationary. The pinion gears 140 being mounted on the spinner plate 126,and meshed with the gear 134, will move along the circumference of nowstationary gear 134 and will rotate about their respective axes untilengagement of the pin 142 at the opposite ehd of the eccentric slot 144will drive the gear 134 with the rotation of the spinner plate 126,limiting the rotation of the pinion gears 140 to 180. The rotation ofthe pinion gears 140 rotates the tube holders 136 and with same, thetubes 150 and the column of blood sample will be rotated. The brakingmust be gradual and not abrupt so that separation of the column from theinner tube wall will not occur. This is accomplished by the frictiondrag applied to plate 126. The column must rotate with the tube wall.

The spinner motor 156 operates to drive the centrifuge head 120 in aclockwise direction for the next cycle of 45 seconds. At the elapse of45 seconds, thenext paddle 180 will have brought around to depress theactuator 184 and cause a second reversal of the spinner motor 156. Thespinner plate 126 again is brought to a momentary halt, and, inreversing direction,,first moves relative to the gear 134 to bring thepin now at the other end of the slot 144, back to the first,

or now opposite end of said slot 144. The pinion gears 140 have thusbeen rotated 180 about their own axes before any appreciable centrifugalforce has been generated.

On completion of the movement of the pin 142 in the slot 144, andengagement of said pin 142 with the spinner plate 126, the spinner plateand the gear 134 are locked for rotation together, now in thecounterclockwise direction for another 45 seconds until the spinnermotor direction is reversed by engagement of the paddle 180" against theactuator 184 of switch 184 depressing same. The pinion gears and hence,the holders 136, tubes and test sample columns therein, again arerotated about their own axes between applications of centrifugal force.

Coupled rotation of the spinner plate 126 and gear 134 is resumed foranother and final 45' second interval. The timer plate 176 iscontinuously rotating during these last described operations, and,accordingly, continues to rotate. Approximately 45 seconds after thelast-mentioned motor reversal, the pin 182 is brought into contact withthe actuator 186' by the continued rotation of the timer disc 176, theactuator 186 is released from its depressed condition. Now, the motor156 is de-energized and the centrifuge head is brought to a halt.

The tubes 150 with their now partially packed red cell layer, are eachcompared with the initial level and a ratio taken which is reflective ofthe sedimentation rate of the sample. It is possible then to subject thetubes and the samples therein to substantially greater G force, such as100 G in a conventional centrifuge so as to fully pack the red cells.The ratio of the fully packed cell level to the partially packed levelis taken. This ratio, the resultant sedimentation rate taken to providewhat can be described as a Zeta Sedimentation Ratio, is independent ofthe effect of hematocrit and is related to the state of health of thesource individual. The term Zeta refers to the Zeta potential betweencells. The Zeta potential to which reference is made is effected by theconcentration of asymmetrical protein molecules in the blood such asfibrinogen, gamma globuin, etc. The Zeta Sedimentation Ratio" in afashion analogous to the sedimentation rate has been found to beindicative of the state of health of the source individual. Unlikesedimentation rate determinations perse which measure rate of red cellfall, the value described here as the-Zeta Sedimentation Ratio or ZSRprovides a determination of the packing factor or closeness of packingof the cells. As a result, the ZSR is independent of the effect ofhematocrit," the relative quantity of redcells in the whole bloodsample.

What it is desired to be secured by Letters Patent of the United Statesis:

1. A method for studying the sedimentation characteristics of wholeblood comprising the steps of:

periodically applying greater than gravity force laterally to agenerally vertically oriented column of test sample of whole blood inplural cycles of predetermined duration, rotating the verticallyoriented column about its own long axis between each cycle, observingthe level of the resulting packed portion of said column of test sampleafter completion of all said cycles and determining the relativeconcentration of cells in the packed and unpacked portions of saidcolumn.

2. The method as claimed in claim 1 wherein the greater than gravityforce is applied to said column in a direction substantially normal tothe long axis of said column.

3. The method as claimed in claim 1 in which the column is definedwithin an elongate tube of capillary proportions and the rotation of thetube about its own long axis is performed so that the column rotateswith the tube wall.

4. The method as claimed in claim 1 in which at least one of said cyclesis of substantially greater duration than the others.

5. The method as claimed in claim 1 wherein the column of test sample isrotated 180 about its own long axis.

6. The method as claimed in claim 1 wherein the rotation of the columnoccurs only when there is less than one G force acting laterallyrelative to the column.

7. The method as claimed in claim 1 wherein the test column istranslated along a circular path at a rate sufficient to apply acentrifugal force greater than one G laterally to the test column inperiodic serial intervals of predetermined duration and the test columnis rotated about its own axis between-said intervals only when saidcentrifugal force acting laterally is less than one G.

8. The method as claimed in claim 7 wherein the rotation of the testcolumn about its own axis is effected by reversal of the direction inwhich the test column is translated along said circular path.

9. The method as claimed in claim 8 wherein the rotation of the testcolumn about its own axis occurs on initiation of the change ofdirection of translation of said test column.

10. The method as claimed in claim 1 and the additional step of fullypacking the column of test sample on completion of all the said cyclesby applying greater than 100 G force to the test column after all thecycles are completed and once the determination of concentration ofcells in the initially packed portion has been completed.

11. The method as claimed in claim 1 wherein said determination is madeby observation of the level of packed cells on completion of all thecycles and comparison of such level to the initial level of said testsample to provide a first packing factor.

12. The method as claimed in claim 11 and the additional steps ofapplying high G force to the test sample column subsequent to completionof said comparison to Pack a to a x m extenmhsre ft r 6992i!!- ing thefinal level of packed cells to the level of the cycled column to obtaina second packing factor and then comparing the pair of packing factorsto provide aratio. V H

13. The method asclairrie d in claim 1 in which the greater than gravityforce is applied in at least two cycles to accelerate the formation ofrouleaux in the sample column, next in a third cycle of a duration atleast twice the duration of each of the first two cycles whereby tocause the formed rouleaux and the plasma 15. The method as claimed inclaim 1 in which the greater than gravity force is applied in at leasttwo ap-.

plications of a first duration to accelerate the formation of rouleauxin the sample column, next in a third application having a durationapproximately three times longer than each of the first two applicationswhereby to cause the formed rouleaux and the plasma components tomigrate one through the other with the formed rouleaux assuming a thinlayer on one side of the column, applying at least one application ofgreater than gravity force for the same duration as each of said firsttwo applications, the column being brought to restso that the rouleauxlayer is permitted to fall to the bottom of the column for observationof level.

16. The method as claimed in claim 1 in which the applied force is ofthe range of from 2 to 12 G.

17. The method as claimed in claim 1 in which the applied force is from5 to 9 G.

18. The method as claimed in claim 1 in which the applied force is from6 to 8 G.

' 19. The method as claimed in claim 1 in which said column is definedby filling an elongate tube with test sample to a predetermined level.orienting said filled tube in a centrifuge head with its long axissubstantially parallel to the axis of rotation of the centrifuge headand rotating said tube in a circumferential path about said rotationalaxis of the centrifuge head during each cycle.

20. The method as claimed in claim 19 in which the greater than gravityforce is applied to the column inclined at an angle theta from thevertical such that the product of the relative centrifugal force appliedand the tangent of the angle theta is a value no higher :than 1.

z 21. The method as claimed in claim 19 in which the column is rotatedfirst in at least two separated but successive cycles of predeterminedduration, each centrifugation cycle being separated by a condition underwhich the column is under less than one G force acting laterallythereto, the column being rotated 180 about its own long axis onlyduring the said condition subsequent to reaching said condition.

22. The method as claimed in claim 21 in which the rotation of thecolumn is effected by reversing the direction of rotation of thecentrifuge head.

23. The method as claimed in claim 1 in which a sample of whole blood isdeposited in an elongate tube to define the column, the tube is placedin a centrifuge oriented substantially parallel to the rotational axisthereof, the column being rotated circumferentially about the centrifugeaxis to apply said greater than gravity force; said circumferentialrotation occurring in cycles of predetermined duration, eachcircumferential rotation being separated by a period when the centrifugeis brought to a substantially at rest condition, and the column isrotated about its own axis only subsequent to reaching saidsubstantially at rest condition.

24. The method as claimed in claim 23 in which the duration of thecycles is inversely prportional to the G force generated by thecentrifuge and applied to the column.

25. The method as claimed in claim 24 in which the orientation of thetest column is no greater than an angle of 6 from vertical orientation.

Poi-1050 I I i I Potent Anomeyf Supplla Dlvlll 7 hand Publhhen, Bayonne,N.3. u7

(5/69) T v I STATES PATENT OFFICE CERTIFICATE OF CORRECTION Patent 110-3' 8.241841 7 Dated -Y 1974 It is certified jrhat error appears in theabove-identified petent and that said Letters Patent are herebycorrected as shown below:

Eolumn 1, line 15, aftef "improved" insert methodcolumn 3,

line 54, change "-"is" to -in--; column 7, line 32, change "comapred" toc.ompa'red-; column 15, line '46, 140 should be --l40 and in heavy type.

Signed anaeealed this 29th day of October 1974.

(SEAL) Attest:

V McCOY M. (mason JR. 'c. MARSHALL DANN Attesting Officer Commissionerof Patents

1. A method for studying the sedimentation characteristics of whole blood comprising the steps of: periodically applying greater than gravity force laterally to a generally vertically oriented column of test sample of whole blood in plural cycles of predetermined duration, rotating the vertically oriented column about its own long axis between each cycle, observing the level of the resulting packed portion of said column of test sample after completion of all said cycles and determining the relative concentration of cells in the packed and unpacked portions of said column.
 2. The method as claimed in claim 1 wherein the greater than gravity force is applied to said column in a direction substantially normal to the long axis of said column.
 3. The method as claimed in claim 1 in which the column is defined within an elongate tube of capillary proportions and the rotation of the tube about its own long axis is performed so that the column rotates with the tube wall.
 4. The method as claimed in claim 1 in which at least one of said cycles is of substantially greater duration than the others.
 5. The method as claimed in claim 1 wherein the column of test sample is rotated 180* about its own long axis.
 6. The method as claimed in claim 1 wherein the rotation of the column occurs only when there is less than one G force acting laterally relative to the column.
 7. The method as claimed in claim 1 wherein the test column is translated along a circular path at a rate sufficient to apply a centrifugal force greater than one G laterally to the test column in periodic serial intervals of predetermined duration and the test column is rotated about its own axis between said intervals only when said centrifugal force acting laterally is less than one G.
 8. The method as claimed in claim 7 wherein the rotation of the test column about its own axis is effected by reversal of the direction in which the test column is translated along said circular path.
 9. The method as claimed in claim 8 wherein the rotation of the test column about its own axis occurs on initiation of the change of direction of translation of said test column.
 10. The method as claimed in claim 1 and the additional step of fully packing the column of test sample on completion of all the said cycles by applying greater than 100 G force to the test column after all the cycles are completed and once the determination of concentration of cells in the initially packed portion has been completed.
 11. The method as claimed in claim 1 wherein said determination is made by observation of the level of packed cells on completion of all the cycles and comparison of such level to the initial level of said test sample to provide a first packing factor.
 12. The method as claimed in claim 11 and the additional steps of applying high G force to the test sample column subsequent to completion of said comparison to pack same to a maximum extent, thereafter comparing the final level of cells to the level of the cycled column to obtain a second packing factor and then comparing the pair of packing factors to provide a ratio.
 13. The method as claimed in claim 1 in which the greater than gravity force is applied in at least two cycles to accelerate the formation of rouleaux in the sample column, next in a third cycle of a duration at least twice the duration of each of the first two cycles whereby to cause the formed rouleaux and the plasma components to migrate one through the other with the formed rouleaux assuming a thin layer on one side of the column, lastly applying at least one cycle of greater than gravity force for the same duration as each of the first two cycles.
 14. The method as claimed in claim 13 in which each cycle is separated by rotation of the test column 180* about its own axis only when the force applied laterally to the column is less than one G.
 15. The method as claimed in claim 1 in which the greater than gravity force is applied in at least two applications of a first duration to accelerate the formation of rouleaux in the sample column, next in a third application having a duration approximately three times longer than each of the first two applications whereby to cause the formed rouleaux and the plasma components to migrate one through the other with the formed rouleaux assuming a thin layer on one side of the column, applying at least one application of greater than gravity force for the same duration as each of said first two applications, the column being brought to rest so that the rouleaux layer is permitted to fall to the bottom of the column for observation of level.
 16. The method as claimed in claim 1 in which the applied force is of the range of from 2 to 12 G.
 17. The method as claimed in claim 1 in which the applied force is from 5 to 9 G.
 18. The method as claimed in claim 1 in which the applied force is from 6 to 8 G.
 19. The method as claimed in claim 1 in which said column is defined by filling an elongate tube with test sample to a predetermined level, orienting said filled tube in a centrifuge head with its long axis substantially parallel to the axis of rotation of the centrifuge head and rotating said tube in a circumferential path about said rotational axis of the centrifuge head during each cycle.
 20. The method as claimed in claim 19 in which the greater than gravity force is applied to the column inclined at an angle theta from the vertical such that the product of the relative centrifugal force applied and the tangent of the angle theta is a value no higher than
 1. 21. The method as claimed in claim 19 in which the column is rotated first in at least two separated but successive cycles of predetermined duration, each centrifugation cycle being separated by a condition under which the column is under less than one G force acting laterally thereto, the column being rotated 180* about its own long axis only during the said condition subsequent to reaching said condition.
 22. The method as claimed in claim 21 in which the rotation of the column is effected by reversing the direction of rotation of the centrifuge head.
 23. The method as claimed in claim 1 in which a sample of whole blood is deposited in an elongate tube to define the column, the tube is placed in a centrifuge oriented substantially parallel to the rotational axis thereof, the column being rotated circumferentially about the centrifuge axis to apply said greater than gravity force, said circumferential rotation occurring in cycles of predetermined duration, each circumferential rotation being separated by a period when the centrifuge is brought to a substantially at rest condition, and the column is rotated about its own axis only subsequent to reaching said substantially at rest condition.
 24. The method as claimed in claim 23 in which the duration of the cycles is inversely prportional to the G force generated by the centrifuge and applied to the column.
 25. The method as claimed in claim 24 in which the orientation of the test column is no greater than an angle of 6* from vertical orientation. 